WO2018120337A1

WO2018120337A1 - Terpene synthase and use thereof

Info

Publication number: WO2018120337A1
Application number: PCT/CN2017/071449
Authority: WO
Inventors: 刘天罡; 卞光凯; 韩以超; 侯安伟; 苑玉杰; 刘然
Original assignee: 武汉臻智生物科技有限公司
Priority date: 2016-12-27
Filing date: 2017-01-17
Publication date: 2018-07-05
Also published as: CN108239631B; CN108239631A

Abstract

Provided are a terpene synthase, a nucleic acid molecule which encodes the terpene synthase, a construct which contains the nucleic acid molecule, a recombinant cell, and a use thereof, as well as a method for synthesizing a terpenoid compound, wherein a catalytic substrate of the terpene synthase is a compound having 10-25 carbon atoms.

Description

Anthraquinone synthase and use thereof

Technical field

The invention relates to the field of biology. In particular, the invention relates to an indole synthase and its use. More specifically, the invention relates to steroid synthase, nucleic acid molecules, constructs, recombinant cells, and uses thereof, and methods of synthesizing terpenoids.

Background technique

Terpenoids are a general term for compounds containing isoprene units. To date, approximately 76,000 terpenoids have been found in animals, plants and microorganisms. It is widely used in the perfume production industry, health care products industry, agricultural production and medical industry.

However, current terpenoids remain to be studied.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art at least to some extent.

To this end, in one aspect of the invention, the invention proposes a terpenoid synthase. According to an embodiment of the invention, the catalytic substrate of the quinone synthase is a compound having 10 to 25 carbon atoms. The indole synthase of the present invention is capable of catalyzing a compound of a long carbon chain and has a broad spectrum and is capable of catalyzing various substrates in order to obtain different terpenoids.

According to an embodiment of the present invention, the above-described steroid synthase may further have the following additional technical features:

According to an embodiment of the present invention, the catalytic substrate is selected from the group consisting of: geranyl pyrophosphate; geranyl pyrophosphate; farnesyl pyrophosphate; geranylgeranyl pyrophosphate; and geranyl method Nicot pyrophosphate. Each of the terpene synthases according to embodiments of the present invention is capable of catalyzing the synthesis of terpenoids from the above substrates, and different terpenoid synthases catalyze the different terpenoids for the same substrate.

According to an embodiment of the present invention, the steroid synthase has the amino acid sequence of any one of SEQ ID NOS: 1 to 6. Thus, the hydrazine synthase according to an embodiment of the present invention has a broad spectrum and is capable of catalyzing various substrates in order to obtain different terpenoids.

In another aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes a steroid synthase as described above. Thus, a nucleic acid molecule according to an embodiment of the invention is capable of efficiently encoding a terpene synthase to catalyze a variety of substrates in order to obtain different terpenoids.

According to an embodiment of the invention, the nucleic acid molecule described above may also have the following additional technical features:

According to an embodiment of the present invention, the nucleic acid molecule has the nucleotide sequence shown in any one of SEQ ID NOS: 7 to 12. Thus, a nucleic acid molecule according to an embodiment of the invention is capable of efficiently encoding a terpene synthase to catalyze a variety of substrates in order to obtain different terpenoids.

In yet another aspect of the invention, the invention proposes a construct. According to an embodiment of the invention, the construct contains a nucleic acid molecule as described above. Thus, a construct according to an embodiment of the invention can encode a plurality of substrates by expressing a nucleic acid molecule encoding a synthetic terpene synthase to obtain different terpenoids.

In yet another aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises: a first nucleic acid molecule encoding a purine synthase. Thus, by culturing the recombinant cells, a hydrazine synthase is obtained, thereby catalyzing various substrates to obtain different terpenoids.

According to an embodiment of the present invention, the steroid synthase has the amino acid sequence of any one of SEQ ID NOS: 1 to 6. Thus, by culturing the recombinant cells, a hydrazine synthase is obtained, thereby catalyzing various substrates to obtain different terpenoids.

According to an embodiment of the present invention, the first nucleic acid molecule has the nucleotide sequence shown in any one of SEQ ID NOS: 7 to 12. Thus, by culturing the recombinant cell, a hydrazine synthase is obtained, thereby catalyzing a plurality of substrates to obtain different hydrazines. Class of compounds.

According to an embodiment of the present invention, the recombinant cell further comprises: a second nucleic acid molecule selected from at least one of the following: an atoB gene derived from Escherichia coli XL1-blue or an idi gene; The erg13 gene, tHMG1 gene, erg12 gene, erg8 gene or mvd1 gene of yeast INVSC1. High yield of terpenoids is achieved by over-expression of the above genes in order to synthesize a large amount of catalytic substrate.

In yet another aspect of the invention, the invention provides the use of a steroid synthase or nucleic acid molecule or construct or recombinant cell described above for the synthesis of a steroid. Thereby, in order to obtain different terpenoids.

According to an embodiment of the present invention, the synthesis is carried out in a host cell, and the catalytic substrate of the steroid synthase is obtained by overexpressing at least one of the following genes in a host cell: from the large intestine The atoB gene or idi gene of Bacillus XL1-blue; the erg13 gene, tHMG1 gene, erg12 gene, erg8 gene or mvd1 gene derived from Saccharomyces cerevisiae INVSC1. High yield of terpenoids is achieved by over-expression of the above genes in order to synthesize a large amount of catalytic substrate.

According to an embodiment of the invention, the terpenoid has a structure of one of the following:

In yet another aspect of the invention, the invention provides a method of synthesizing the above described terpenoids. According to an embodiment of the present invention, the method comprises: cultivating the recombinant cells described above under conditions suitable for expression of the terpenoid to obtain a culture product; and isolating the terpenoid from the culture product . Thus, in order to obtain a large number of different terpenoids.

The additional aspects and advantages of the invention will be set forth in part in the description which follows.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

1 shows a GC-MS detection FgMS in vitro reaction chromatogram according to an embodiment of the present invention;

2 shows a chromatogram of in vitro reaction of FgGS detected by GC-MS according to an embodiment of the present invention;

Figure 3 shows a schematic diagram of the structure of plasmid pMH1 according to one embodiment of the present invention;

Figure 4 is a schematic view showing the structure of plasmid pFZ81 according to an embodiment of the present invention;

Figure 5 is a schematic view showing the structure of plasmid pGB309 according to an embodiment of the present invention;

Figure 6 shows a schematic diagram of the structure of plasmid pGB310 according to one embodiment of the present invention;

Figure 7 is a schematic view showing the structure of plasmid pGB311 according to an embodiment of the present invention;

Figure 8 is a schematic view showing the structure of plasmid pGB312 according to an embodiment of the present invention;

Figure 9 is a schematic view showing the structure of plasmid pGB313 according to an embodiment of the present invention;

Figure 10 is a schematic view showing the structure of plasmid pGB314 according to an embodiment of the present invention;

Figure 11 is a schematic view showing the structure of plasmid pGB147 according to an embodiment of the present invention;

Figure 12 is a schematic view showing the construction of a plasmid and a mutant strain for synthesizing different types of products for FgMS and FgGS fermentation according to an embodiment of the present invention;

Figure 13 is a schematic view showing a method for synthesizing a terpenoid compound according to an embodiment of the present invention, wherein (a) is a schematic diagram of a highly efficient synthesis of a terpenoid chassis of the MVA pathway; (b) is a purine synthase with different chain lengths a schematic diagram of a combination of olefin pyrophosphate synthase, a compound of sesquiterpene (C15), diterpene (C20), and a sesquiterpene (C25); (c) a fermentation product of six E. coli strains (T7-T12) GS-MS chromatogram

Figure 14 shows a mass spectrum of FgMS and FgGS fermentation products according to one embodiment of the present invention;

Figure 15 shows the product of AaTS synthesis in accordance with one embodiment of the present invention;

Figure 16 shows a spectrum of compound (1) according to one embodiment of the present invention, wherein a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400MHz); c is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101MHz); d is the HSQC spectrum (CDCl ₃ ); e is the ¹ H- ¹ H COSY spectrum (CDCl ₃ ); f is the HMBC spectrum (CDCl ₃ );

Figure 17 shows a spectrum of compound (2) according to one embodiment of the present invention, wherein a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400MHz); c is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101MHz); d is the HSQC spectrum (CDCl ₃ ); e is the ¹ H- ¹ H COSY spectrum (CDCl ₃ ); f is the HMBC spectrum (CDCl ₃ );

Figure 18 shows a spectrum of compound (3) according to one embodiment of the present invention, wherein a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400MHz); c is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101MHz); d is the HSQC spectrum (CDCl ₃ ); e is the ¹ H- ¹ H COSY spectrum (CDCl ₃ ); f is the HMBC spectrum (CDCl ₃ );

Figure 19 shows a spectrum of a compound (4) according to one embodiment of the present invention, wherein a is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400 MHz); b is a carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101 MHz) );

Figure 20 shows a spectrum of compound (5) according to one embodiment of the present invention, wherein a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400MHz); c is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101MHz); d is the HSQC spectrum (CDCl ₃ ); e is the ¹ H- ¹ H COSY spectrum (CDCl ₃ ); f is the HMBC spectrum (CDCl ₃ );

Figure 21 shows a spectrum of the compound (6) according to one embodiment of the present invention, wherein a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400MHz); c is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101MHz); d is the HSQC spectrum (CDCl ₃ ); e is the ¹ H- ¹ H COSY spectrum (CDCl ₃ ); f is the HMBC spectrum (CDCl _3);

Figure 22 shows a spectrum of the compound (7) according to one embodiment of the present invention, wherein a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400MHz); c is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101MHz); d is the HSQC spectrum (CDCl ₃ ); e is the ¹ H- ¹ H COSY spectrum (CDCl ₃ ); f is the HMBC spectrum (CDCl ₃ );

Figure 23 shows a spectrum of compound (8) according to one embodiment of the present invention, wherein a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400MHz); c is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101MHz); d is the HSQC spectrum (CDCl ₃ ); e is the ¹ H- ¹ H COSY spectrum (CDCl ₃ ); f is the HMBC spectrum (CDCl ₃ );

Figure 24 shows a spectrum of compound (9) according to one embodiment of the present invention, wherein a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400MHz); c is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101MHz); d is the HSQC spectrum (CDCl ₃ ); e is the ¹ H- ¹ H COSY spectrum (CDCl ₃ ); f is the HMBC spectrum (CDCl ₃ );

Figure 25 shows a spectrum of compound (10) according to one embodiment of the present invention, wherein a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl _3, 400MHz); c is carbon spectra ^{_{(13 C NMR, CDCl 3,}} 101MHz); d is a HSQC spectrum (CDCl _3); e is a spectrum of ^¹ H- ¹ H ^COSY (CDCl _3); f is the HMBC spectrum Figure (CDCl ₃ );

Figure 26 shows a spectrum of the compound (11) according to one embodiment of the present invention, wherein a is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400 MHz); b is a carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101 MHz) );

Figure 27 shows a spectrum of the compound (12) according to one embodiment of the present invention, wherein a is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ , 400 MHz); b is a carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101 MHz) );

28 shows a spectrum of a compound (53) in which a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ ) according to an embodiment of the present invention. , 400MHz); c is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101MHz); d is the HSQC spectrum (CDCl ₃ ); e is the ¹ H- ¹ H COSY spectrum (CDCl ₃ ); f is the HMBC spectrum (CDCl _3); and

29 shows a spectrum of a compound (54) in which a is a planar structure and ¹ H- ¹ H COSY and a critical HMBC correlation; b is a hydrogen spectrum ( ¹ H NMR, CDCl ₃ ) according to an embodiment of the present invention. , 400MHz); c is the carbon spectrum ( ¹³ C NMR, CDCl ₃ , 101MHz); d is the DEPT 135° spectrum (CDCl ₃ ); e is the HSQC spectrum (CDCl ₃ ); f is ¹ H- ¹ H COSY Spectrum (CDCl ₃ ); g is the HMBC spectrum (CDCl ₃ ).

detailed description

Embodiments of the present invention are described in detail below. The embodiments described below are illustrative only and are not to be construed as limiting the invention.

Purine synthase

In one aspect of the invention, the invention provides a steroid synthase. According to an embodiment of the invention, the catalytic substrate of the steroid synthase is a compound having 10 to 25 carbon atoms. The inventors have found that most of the existing terpenoid synthase can only catalyze the substrate of short carbon chain (for example, 5 to 10 carbon atoms), and the specificity is strong, and only a specific substrate can be catalyzed, and the corresponding axene can be obtained. Compound. The hydrazine synthase of the present invention is not only capable of catalyzing compounds of short carbon chains, such as isopentenyl pyrophosphate and olefins. Propyl pyrophosphate, which is also capable of catalyzing long carbon chain compounds (e.g., 10 to 25 carbon atoms), has a broad spectrum and is capable of catalyzing a variety of substrates to obtain different terpenoids.

According to an embodiment of the invention, the catalytic substrate is selected from one of the following: geranyl pyrophosphate (GPP); farnesyl pyrophosphate (FPP); geranylgeranyl pyrophosphate (GGPP); and geranyl Farnesyl pyrophosphate (GFPP). Each of the terpene synthases of the present invention is capable of catalyzing the synthesis of terpenoids from the above substrates, and different terpenoid synthases catalyze the different terpenoids for the same substrate.

According to an embodiment of the present invention, the steroid synthase has the amino acid sequence of any one of SEQ ID NOS: 1 to 6.

The inventors have found that the above-mentioned steroidal synthase is provided with farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), geranyl farnesyl pyrophosphate (GFPP), and isopentenyl pyrophosphate ( IPP), allyl pyrophosphate (DMAPP) and geranyl pyrophosphate (GPP) are converted to terpenoid functions. Further, the terpene synthase can efficiently obtain a plurality of different terpenoids by catalyzing a substrate.

Indole synthase 1 (abbreviated as FgMS) has the amino acid sequence shown in SEQ ID NO: 1, as follows:

Indole synthase 2 (abbreviated as D510A) has the amino acid sequence shown in SEQ ID NO: 2, as follows:

Indole synthase 3 (abbreviated as FgGS) has the amino acid sequence shown in SEQ ID NO: 3, as follows:

Indole synthase 4 (GGPPS-Aa for short) has the amino acid sequence shown in SEQ ID NO: 4, as follows:

Indole synthase 5 (abbreviated as AaTS) has the amino acid sequence shown in SEQ ID NO: 5, as follows:

Indole synthase 6 (abbreviated as FgAS) has the amino acid sequence shown in SEQ ID NO: 6, as follows:

According to an embodiment of the present invention, FgMS, D510A, FgGS and FgAS are isolated from Fusarium graminearum of Taxus, and GGPPS-Aa and AaTS are isolated from Alternaria alternata of Taxus.

According to an embodiment of the present invention, by homologous alignment of FgMS, D510A, FgGS, GGPPS-Aa, FgAS and AaTS in the NCBI database, the serial number is found to be XP_018034954.1, AHY23929.1, XP_002846409.1, XP_003025181. 1, XP_003236661.1, CEF73922.1, XP_009262810.1, OBS27829.1.1, XP_011317573.1, EYB24413.1, XP_011317623.1, OBS27869.1, XP_009262762.1, XP_003343918.1, KFA74407.1 and FgMS, D510A, FgGS, FgAS, GGPPS-Aa and AaTS have high homology, and it is speculated that the above sequence also has a broad spectrum of steroid synthase properties, which can catalyze a variety of substrates to obtain different terpenoids.

Nucleic acid molecule

In another aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes a preceding purine synthase. Thus, a nucleic acid molecule according to an embodiment of the present invention is capable of efficiently encoding a terpene synthase, and the obtained purine synthase has a broad spectrum and is capable of catalyzing various substrates to obtain different terpenoids.

Nucleic acid molecule 1 has the nucleotide sequence shown in SEQ ID NO: 7, and is capable of encoding FgMS, a specific nucleotide sequence. as follows:

Nucleic acid molecule 2 has the nucleotide sequence shown in SEQ ID NO: 8, encoding D510A, and the specific nucleotide sequence is as follows:

Nucleic acid molecule 3 has the nucleotide sequence shown in SEQ ID NO: 9, encoding FgGS, and the specific nucleotide sequence is as follows:

The nucleic acid molecule 4 has the nucleotide sequence shown in SEQ ID NO: 10 and encodes GGPPS-Aa, and the specific nucleotide sequence is as follows:

Nucleic acid molecule 5 has the nucleotide sequence shown in SEQ ID NO: 11, encoding AaTS, and the specific nucleotide sequence is as follows:

The nucleic acid molecule 6 has the nucleotide sequence shown in SEQ ID NO: 12 and encodes FgAS. The specific nucleotide sequence is as follows:

Those skilled in the art will appreciate that the features and advantages previously described for the hydrazine synthase are equally applicable to the nucleic acid molecule and will not be described herein.

Construct

In yet another aspect of the invention, the invention proposes a construct. According to an embodiment of the invention, the construct comprises a nucleic acid molecule as described above. Thus, a construct according to an embodiment of the invention can encode a plurality of substrates by expressing a nucleic acid molecule encoding a synthetic terpene synthase to obtain different terpenoids.

Those skilled in the art will appreciate that the features and advantages previously described for nucleic acid molecules are equally applicable to such constructs and will not be described herein.

Recombinant cell

In yet another aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises: a first nucleic acid molecule, the first nucleic acid molecule encoding a purine synthase. Thus, by culturing the recombinant cells, a hydrazine synthase is obtained, thereby catalyzing various substrates to obtain different terpenoids.

According to an embodiment of the present invention, the steroid synthase has the amino acid sequence of any one of SEQ ID NOS: 1 to 6, and according to another embodiment of the present invention, the first nucleic acid molecule has SEQ ID NOs: 7 to 12 A nucleotide sequence as shown.

According to an embodiment of the present invention, further comprising: a second nucleic acid molecule selected from at least one of the following: an atoB gene derived from Escherichia coli XL1-blue (acetoacetyl-CoA thioesterase), an idi gene ( Isopentenyl pyrophosphate isomerase) gene; erg13 (HMG-CoA synthase) gene derived from Saccharomyces cerevisiae INVSC1, tHMG1 gene (HMG-CoA reductase, deleted transmembrane region of HMG1), erg12 gene (metholone) Acid kinase), erg8 gene (mevalonate-5-phosphate kinase), mvd1 gene (mevalonate-5-pyrophosphate kinase).

The inventors have found that the above genes are closely related to the catalytic substrate of the steroid synthase, and a large amount of the catalytic substrate can be obtained by overexpressing the above gene. Further, under the catalysis of a hydrazine synthase, a large amount of a quinone compound having a small amount of synthesis can be synthesized in a large amount.

Those skilled in the art will appreciate that the features and advantages previously described for steroid synthase and nucleic acid molecules are equally applicable to the recombinant cell and will not be described herein.

Use of recombinant cells in the synthesis of terpenoids

In yet another aspect of the invention, the invention provides the use of a steroid synthase or nucleic acid molecule or construct or recombinant cell described above for the synthesis of a steroid. Thus, by culturing the recombinant cells, a hydrazine synthase is obtained, thereby catalyzing various substrates to obtain different terpenoids.

According to an embodiment of the present invention, the synthesis is carried out in a host cell, and the catalytic substrate of the steroid synthase is obtained by overexpressing at least one of the following genes in the host cell: derived from Escherichia coli XL1-blue atoB gene or idi gene; erg13 gene, tHMG1 gene, erg12 gene, erg8 gene or mvd1 gene derived from Saccharomyces cerevisiae INVSC1. A large amount of substrate is obtained by overexpressing the above genes. Further, under the catalysis of a hydrazine synthase, a large amount of a quinone compound having a small amount of synthesis can be synthesized in a large amount.

It should be noted that since the indole synthase of the present invention has a broad spectrum, it is possible to catalyze different substrates to obtain different terpenoids. According to an embodiment of the present invention, the quinone compound has a structure of one of the following. The inventors have found that new terpenoids, such as compounds (5), (6), (7), (8), (9), (10), have been obtained under the catalytic action of the hydrazine synthase described above. (11), (12) and (54).

Those skilled in the art will appreciate that the features and advantages previously described for steroid synthase, nucleic acid molecules, constructs, and recombinant cells are equally applicable to this use and will not be described herein.

Method for synthesizing terpenoids

In yet another aspect of the invention, the invention provides a method of synthesizing the above described terpenoids. According to an embodiment of the present invention, the method comprises: culturing the preceding recombinant cells under conditions suitable for expression of the terpenoid to obtain a culture product; and isolating the terpenoid from the culture product. Thus, in order to obtain a large number of different terpenoids.

Those skilled in the art will appreciate that the features and advantages previously described for recombinant cells are equally applicable to the method of synthesizing terpenoids and will not be described herein.

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in the art or in accordance with the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

Example 1 In vitro verification of the function of steroid synthase

(1) Purification of protein

The expression vector containing the gene of interest (the nucleotide sequence shown in any one of SEQ ID NOS: 7 to 12) is transformed into the expression host E. coli BL21 (DE3), and after transformation, the monoclonal antibody is picked up to the LB culture containing the corresponding antibiotic. Base, 37 ° C, 220 rpm overnight culture. Transfer to 1 L of fresh LB medium containing the corresponding antibiotics at 1% inoculum, incubate at 220 ° C, rpm to OD _{600 of} about 0.6-0.8 at 37 ° C, cool down to 16 ° C, add IPTG at a final concentration of 0.1 mM, 16 ° C Incubate at 220 rpm for 16-18 h. The cells were collected by centrifugation at 8 000 rpm for 5 min, and then the cells were completely resuspended with 30-40 mL of Protein Purification Buffer A (Buffer A: 50 mM Tris-HCl, 300 mM NaCl, 4 mM β-mercaptoethanol, pH 7.6), sonicated (pulse 5 s, pause for 8 s). , ultrasonic disruption 5min). Centrifuge at 12,000 g for 30 min at 4 ° C, collect the supernatant, centrifuge at 20,000 rpm for 1 h at 4 ° C, collect the supernatant, filter with a 0.45 μm filter, and add 6% buffer B (Buffer B: 500 mM imidazole added to Buffer A) The imidazole was about 30 mM and mixed for later use.

The histidine-tagged protein was purified using Bio-Rad's Biologic DuoFlow Chromatography System. The protein separation column was loaded onto the FPLC for control. The flow rate of the FPLC was always 1.5 mL/min, and the flow rate of the sample was automatically loaded at 2 mL/min. The obtained supernatant sample was purified by Biorad using a 5 mL Hitrap HP Ni-NTA column, which was first passed through 30 mL (6 column volumes) of buffer A (Buffer A: 50 mM Tris-HCl, 300 mM). Balance of NaCl, 4 mM β-mercaptoethanol, pH 7.6, then load the prepared 30 mL supernatant into the autosampler to On the column, the column was washed with 20 mL of buffer A (4 column volumes), at which time a linear gradient of buffer B (50 mM Tris-HCl, 150 mM NaCl, 250 mM Imidazole pH 7.6) was initiated at 100 mL (20 Within the flow rate of column volume, buffer B was increased from 0% to 100%, and the column was washed with 20 mL (4 column volumes) of 100% buffer B. The histone-tagged protein of interest was collected by UV absorption and detected by SDS-PAGE. The purely pure fractions were collected and concentrated by centrifugation to 2.5 mL by Millipore's centrifugal concentrator Amicon Centricon-10 (molecular weight below 10,000), then desalted and exchanged by Pharmacia PD-10 column. To buffer C (20 mM Tris-HCl, 10 mM NaCl, pH 7.6).

The volume of protein from the PD-10 column was diluted to 3.5 mL, and the sample was loaded onto an ion exchange column Hitrap 16/10Q/FF and purified by FPLC. The ion exchange column was flushed with buffer C (20 mL (1 column volume) after completion of sample loading, and then started to elute with buffer D (20 mM Tris-HCl, 1 M NaCl, pH 7.6) at a flow rate of 20 mL. In the buffer D increased from 0% to 30%; after the flow rate of 40mL (2 column volume), the buffer D increased from 30% to 50%; then the flow rate of 20mL (1 column volume), buffer D by 50% increase to 100%; finally use 100% 20mL buffer D to clean the column. The eluted protein of interest was collected by UV absorption and detected by SDS-PAGE.

The resulting protein was further concentrated by centrifugation to 2 mL and loaded onto a gel filtration column (gel filtration Superdex 200 column) using buffer E (50 mM phosphate buffer containing 10% glycerol) on FPLC before loading. , pH 7.6) equilibrated 240 mL (2 column volumes) and the eluted proteins of interest (FgMS, D510A, FgGS, GGPPS-Aa and AaTS) were recovered according to UV absorption. The sample was concentrated to 2 mL with a protein centrifugation concentrating column, and the liquid nitrogen was quickly frozen after storage and stored in a -80 ° C refrigerator.

(2) In vitro enzymatic reaction of terpenoid synthase

In order to fully understand the potential of FgMS and FgGS to catalyze the synthesis of the corresponding products from different substrates, the following in vitro enzymatic reaction system was established: 10 μM of purified protein was added to 200 μL of a final concentration of 50 mM PB buffer (pH 7.6) buffer containing 10% glycerol. 100 μM of substrate (GPP, FPP, GGPP or GFPP) and 2 mM Mg ^{2+ were} reacted overnight at 30 °C. It was then extracted twice with an equal volume of n-hexane, the organic phases were combined and the resulting product was detected by GC-MS.

The GC-MS used for the detection of terpenoids was a Thermo TRACE GC ULTRA gas chromatograph equipped with TSQ QUANTUM XLS MS and the gas chromatographic column was TRACE TR-5MS (30 m x 0.25 mm x 0.25 um). 1 μL of each injection was injected, and high-purity helium was used as a carrier gas, and the flow rate was set to 1 mL/min. The GC conditions were maintained at 80 ° C for 1 min, then ramped to 220 ° C at a rate of 10 ° C/min and maintained at 220 ° C for 15 min. The injector and transfer line temperatures were set to 230 ° C and 240 ° C, respectively.

The results show that both FgMS and FgGS enzymes have a very broad substrate specificity. They are capable of synthesizing the corresponding monoterpenes, sesquiterpenes, diterpenes, and diquinones using four substrates of GPP, FPP, GGPP, and GFPP (Fig. 1, Fig. 2). This is the most widely used terpenoid synthase found in the substrate to date.

Example 2 Construction of an expression vector

Escherichia coli XL1-blue genomic DNA and S. cerevisiae INVSC1 genomic DNA were obtained by purification using Qiagen's Blood and Cell Culture DNA Mini Kit.

Plasmid pMH1 contains the first three genes of the mevalonate pathway: the atoB gene derived from E. coli XL1-blue (acetoacetyl-CoA thioesterase, https://www.ncbi.nlm.nih.gov/nucleotide/313848522? Report=genbank&log$=nuclalign&blast_rank=74&RID=57CNRDHR014&from=2216464&to=2217648), erg13 gene derived from Saccharomyces cerevisiae INVSC1 (HMG-CoA synthase, https://www.ncbi.nlm.nih.gov/nucleotide/1095459859?report= Genbank&log$=nuclalign&blast_rank=1&RID=57DUY RMK014&from=13093&to=14568) and the tHMG1 gene (HMG-CoA reductase, deleted the transmembrane region of HMG1, https://www.ncbi.nlm.nih.gov/nucleotide/1034554135? Report=genbank&log$=nuclalign&blast_rank=1&RID=57DN4VA2015&from=109257&to=110762).

Plasmid pFZ81 contains four genes after the mevalonate pathway: erg12 gene derived from Saccharomyces cerevisiae INVSC1 (mevalonate kinase, https://www.ncbi.nlm.nih.gov/nucleotide/1039023426?report=genbank&log$ = Nuclalign&blast_rank=1&RID=57DY1S95015&from=684102&to=685433), erg8 gene (mevalonate-5-phosphate kinase, https://www.ncbi.nlm.nih.gov/nucleotide/767197525?report=genbank&log$=nuclalign&blast_rank=1&RID =57E0JFF3015&from=684167&to=685522) and the mvd1 gene (mevalonate-5-pyrophosphate kinase, https://www.ncbi.nlm.nih.gov/nucleotide/1034554153?report=genbank&log$=nuclalign&blast_rank=1&RID=57E3RE8N015&from =677640&to=678830), the idi gene derived from E. coli XL1-blue (prenyl pyrophosphate isomerase, https://www.ncbi.nlm.nih.gov/nucleotide/1114169151?report=genbank&log$=nuclalign&blast_rank =1&RID=57E42G K1014&from=3614163&to=3614711).

Plasmid pGB309 contains three genes for the synthesis of a sesquiterpene compound, which is a FgMS gene derived from Fusarium graminearum (SEQ ID NO. 7), the amino acid sequence of which is SEQ ID NO. The idi gene of Bacillus XL1-blue; the FPPS gene derived from geobacillus stearothermophilus (farnesene pyrophosphate synthase, https://www.ncbi.nlm.nih.gov/nucleotide/391609? Report=genbank&log$=nuclalign&blast_rank=1&RID=57EBD7Z0015&from=85&to=978), the gene was subjected to site-directed mutagenesis to obtain the nucleotide sequence shown in SEQ ID NO: 13, which was given GFPPS (Ferrophyllinyl pyrophosphate synthase) The function of synthesizing C25 pyrophosphate from IPP and DMAPP as a substrate for the synthesis of di-half-quinone products.

The nucleotide sequence shown in SEQ ID NO: 13:

Plasmid pGB310 contains three genes for the synthesis of diterpenoids, respectively FgMS derived from Fusarium graminearum and subjected to site-directed mutagenesis (amino acid D mutation at position 510 to A), abbreviated as D510A (SEQ. ID NO.8), whose amino acid sequence is SEQ ID NO. 2; Idi gene derived from Escherichia coli XL1-blue; geranylgeranyl pyrophosphate synthase GGPPS derived from Taxus canadensis (https://www.ncbi.nlm.nih.gov/nucleotide/507118460?report=genbank&log$=nuclalign&blast_rank=1&RID=57EEP8PR014&from=1&to=889), capable of synthesizing geranylgeranyl base with IPP and DMAPP as substrates Pyrophosphoric acid for the synthesis of diterpene products.

Plasmid pGB311 contains three genes for the synthesis of sesquiterpene compounds, respectively D510A derived from Fusarium graminearum and subjected to site-directed mutagenesis of the deletion strand extension domain, abbreviated as D510A; idi gene derived from Escherichia coli XL1-blue; FPPS derived from E. coli XL1-blue can synthesize farnesyl pyrophosphate using IPP and DMAPP as substrates for the synthesis of sesquiterpene.

The plasmid pGB312 contains three genes for synthesizing a sesquiterpene compound, which is a FgGS gene derived from Fusarium graminearum (SEQ ID NO. 9), and its amino acid sequence is SEQ ID NO. 3; The idi gene of Enterobacter XL1-blue; FPPS (farnesene pyrophosphate synthase) derived from geobacillus stearothermophilus, which is subjected to site-directed mutagenesis to obtain the nucleotide represented by SEQ ID NO: The sequence, which is given the function of GFPPS (geranyl farnesyl pyrophosphate synthase), synthesizes the product of C25 pyrophosphate using IPP and DMAPP as a substrate for the synthesis of the sesquiterpene product.

Plasmid pGB313 contains three genes for the synthesis of diterpenoids, which are the FgGS gene derived from Fusarium graminearum; the idi gene derived from Escherichia coli XL1-blue; and the yak based on Taxus canadensis. The geranyl pyrophosphate synthase GGPPS is capable of synthesizing geranylgeranyl pyrophosphate using IPP and DMAPP as a substrate for the synthesis of diterpene products.

Plasmid pGB314 contains three genes for the synthesis of sesquiterpene compounds, namely FgGS derived from Fusarium graminearum; idi gene derived from Escherichia coli XL1-blue; FPPS derived from Escherichia coli XL1-blue, capable of IPP and DMAPP Synthesis of farnesyl pyrophosphate as a substrate for the synthesis of sesquiterpene.

All genes were obtained by PCR amplification, and the primers used are shown in Table 1.

Table 1 primer sequence table

The specific construction method is as follows:

1 Construction of plasmid pMH1

The replicon of the pBBR1MCS plasmid was first replaced with the p15A replicon derived from the pMSD15 plasmid. Plasmid pBBR1MCS was used as template for amplification with primer P1/P2, and p15A replicon was amplified with primer P3/P4. After purification by PCR product, DNA concentration was determined by Nanodrop, then 20 ng pCR amplified p15A fragment and equimolar pBBR1MCS fragment were determined. After mixing, after one round of PCR amplification, the amplification conditions were: 98 ° C, 2 min pre-denaturation, then 30 PCR cycles 98 ° C, 20 s; 60 ° C, 20 s; 72 ° C, 6 min, and finally 72 ° C fully extended for 10 min. Subsequently, E. coli XL1-blue was transformed to obtain plasmid pBBR1MCS/p15A.

The pMH1 plasmid backbone was amplified with the primer P5/P6 using pBBR1MCS/p15A as a template, and the corresponding genes were amplified using P7/P8, P9/P10 and P11/P12 as primers. After purification of the PCR product, 50 ng of pBBR1MCS/p15A amplification product and equimolar amplification products of each gene were mixed, and the volume was adjusted to 5 μL with deionized water, then added to 15 μL of Gibson buffer and mixed, and reacted at 50 ° C for 1 h. After transformation of E. coli XL1-blue, clones were picked and positive clones were sequenced to obtain plasmid pMH1 (Fig. 3).

2 Construction of plasmid pFZ81

The pFZ81 plasmid backbone was amplified with primers P13/P14 using pBBR1MCS-2 as a template, and the corresponding genes were amplified using P15/P16, P17/P18, P19/P20 and P21/P22 as primers. After purification of the PCR product, 50 ng of pBBR1MCS-2 amplification product and equimolar amplification products of each gene were mixed, and the volume was adjusted to 5 μL with deionized water, then added to 15 μL of Gibson buffer and mixed, and reacted at 50 ° C for 1 h. After transformation of E. coli XL1-blue, clones were picked and positive clones were sequenced to obtain plasmid pFZ81 (Fig. 4).

Construction of 3 plasmid pGB309

In order to construct a plasmid producing diploid sputum, we amplified FgMS (F. graminearum mangicdiene synthase), GFPPS (SEQ ID NO. 13) and pGB307 scaffold with primers P33/P39, P40/P41 and P37/P38, respectively. The above three fragments were assembled to obtain plasmid pGB309 (Fig. 5).

Construction of 4 plasmid pGB310

Using the codon-optimized FgMS as a template, F510 fragments of D510A site-directed mutagenesis were amplified by P23/P26 and P24/P25, respectively, and the two fragments were ligated by SOE-PCR to obtain the mutated D510A, which was subsequently cloned into Plasmid pGB302 was obtained on the pET21a(+) plasmid. The idi gene was cloned into pETduet-1 to obtain plasmid pGB307. The D510A, GGPPS and plasmid pGB307 backbones were amplified with primers P33/P34, P35/P36 and P37/P38, and the above three fragments were assembled to obtain plasmid pGB310 (Fig. 6).

Construction of 5 plasmid pGB311

The fpps gene and the idi gene were amplified from the E. coli BL21 (DE3) genome by primers P29/P30 and P31/P32, respectively, and then the fpps gene was cloned into pET21a to obtain plasmid pGB305; idi was cloned into pET21a (+) to obtain plasmid pGB306. . Plasmids pGB305 and pGB306 were digested with XbaI/XhoI, SpeI/XhoI, respectively, and then the idi fragment digested with pGB306 was ligated to plasmid pGB305 by means of homologous enzyme to obtain plasmid pGB308. Subsequently, the fpps-idi fragment was digested with pba308 by XbaI/XhoI and ligated to plasmid pGB302 by homologous enzyme, respectively, to obtain plasmid pGB311 (Fig. 7).

Construction of 6 plasmid pGB312

FgGS (F. graminearum GJ1012 synthase), GFPPS and pGB307 plasmid backbone were amplified with primers P42/46, P41/P47 and P37/P45, and the above three fragments were assembled to obtain plasmid pGB312 (Fig. 8).

Construction of 7 plasmid pGB313

The FgGS, GGPPS and pGB307 plasmid backbones were amplified with primers P42/43, P44/P36 and P37/P45, and the above three fragments were assembled to obtain plasmid pGB313 (Fig. 9).

Construction of 8 plasmid pGB314

The FgGS gene was digested with SacI/HindIII from plasmid pUC57-FgGS (pGB303) containing codon-optimized FgGS and ligated into pET21a(+) to obtain plasmid pGB304. The fpps-idi fragment was subsequently digested from pGB308 by XbaI/XhoI and ligated to plasmid pGB304 by homologous enzymes, respectively, to obtain plasmid pGB314 (Fig. 10).

Example 3 Synthesis of FgMS-derived sesquiterpene compound

In order to produce a sesquiterpene compound, the two plasmids pMH1 and pFZ81 of the mevalonate pathway are simultaneously transferred into the large intestine. BL21(DE3)/pMH1/pFZ81 was obtained in Bacillus sp. BL21 (DE3) and designated as PS, and then pGB309 was transformed into strain PS to obtain strain T7 (Fig. 12).

Subsequently, single clones were picked into 10 mL of LB medium (containing 100 μg/mL ampicillin, 50 μg/mL kanamycin and 34 μg/mL chloramphenicol), cultured at 37 ° C, 220 rpm overnight, followed by 1% inoculation. The amount was inoculated into fresh same medium, and the culture was continued at 37 ° C, 220 rpm until the OD600 was about 0.6-0.8, the temperature was lowered to 16 ° C, and the expression was induced by adding IPTG at a final concentration of 0.1 mM, and the expression was induced to increase to 28 ° C after 18 hours. Fermentation was carried out for 72 h, followed by extraction twice with an equal volume of n-hexane, and the methanol was reconstituted after distillation under reduced pressure for product purification.

The results showed that the mutant strain E.coli T7 containing FgMS was able to synthesize eight sesquiterpenoids using GFPP as a substrate (Fig. 13c, Fig. 14). We successfully purified and identified compound (1) and compound (2). They are two new haploin compounds. Compound (1) has the anti-inflammatory effect of the compounds mangicol A and the precursor of mangicol B. The compound (2) is a precursor substance having a sesquiterpene compound variecolin which inhibits an angiotensin II receptor and has an immunosuppressive effect. In addition, by aligning the GC-MS data of the known sesquiterpene compounds (Fig. 13c, Fig. 14), we found that the compound (37-42) synthesized by the mutant strain E.coli T7 containing FgMS is potential. New terpenoids.

Example 4 Synthesis of FgMS-derived Diterpenoids

To produce the diterpenoid compound, the two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transferred into E. coli BL21 (DE3) to obtain BL21(DE3)/pMH1/pFZ81, designated PS, and then pGB310 was transformed into the strain PS. In the strain T8 was obtained (Fig. 12). Subsequently, fermentation and product extraction were carried out in the same manner as in Example 3.

The results showed that the mutant strain E. coli T8 containing FgMS was able to synthesize ten diterpenoid compounds using GGPP as a substrate (Fig. 13c, Fig. 14). The main product 4 is identical to the compound synthesized by Streptomyces-derived DtcycB reported in the literature, and is a 14-member macrocyclic compound cembrene A (Meguro A, Tomita T, Nishiyama M, et al. Identification and characterization of bacterial diterpene cyclases). That synthesize the cembrane skeleton [J]. ChemBioChem, 2013, 14(3): 316-321.). In addition, a database search (NIST) of some diterpenoid byproducts synthesized by the mutant showed that the compound (18) and the compound (19) contained a skeleton similar to cembrene A, possibly different stereoisomers thereof ( Figure 14). This is the first time we have found a macrocyclic diterpenoid of this type in a fungal-derived terpene synthase. One of the compounds was successfully purified and identified, and the compound (4) was Cembrebe A. Among the remaining nine diterpenoids, the NIST search results of the compound (16-19) showed that the compound (16) was Trachylobane, the compound (18) was Cyclotetradecatetraene, and the compound (17) was E, E-7, 11 , 15-Trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene, compound (19) is (3E,7E,11E)-1-Isopropyl-4,8,12-trimethylcyclotetradeca-3,7, 11-trienol. The remaining structure is unknown for compounds 32-36.

Example 5 Synthesis of FgMS-derived monoterpenoids and sesquiterpene compounds

In order to produce monoterpenes and sesquiterpene compounds, GPP was synthesized based on FPPS with IPP and DMAPP as substrates, and then 1 molecule of IPP was used to generate FPP, and the two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transferred into the large intestine. BL21(DE3)/pMH1/pFZ81 was obtained in Bacillus sp. BL21 (DE3) and designated as PS, and then pGB311 was transformed into strain PS to obtain strain T9 (Fig. 12). Subsequently, fermentation and product extraction were carried out in the same manner as in Example 3.

The results showed that the mutant strain E.coli T9 containing FgMS was able to synthesize 15 sesquiterpene compounds and two monoterpene compounds using FPP as a substrate (Fig. 13c, Fig. 14), in which two monoterpene compounds were compounds (13). And (14). Compound (11) is a linear trans-nerolidol; compound (12) is 2E, 6E-farnesol (2E, 6E-farnesol); NIST search results show that compound (15) is a-Farnesene; compound (20-31) is an unknown structural sesquiterpene compound. The structures of the compounds (13) and (14) were determined by NIST search and comparison with corresponding standards.

At the same time, we also detected two monoterpenoids, linalool and terpineol, in the fermentation product, which is due to the fact that FPPS will first synthesize intermediate GPP in the process of synthesizing FPP.

Example 6 Synthesis of FgGS-derived sesquiterpene compounds

In order to produce a sesquiterpene compound, the two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transferred into E. coli BL21 (DE3) to obtain BL21(DE3)/pMH1/pFZ81, designated PS, and then pGB312 was transformed into In strain PS, strain T10 was obtained (Fig. 12). Subsequently, fermentation and product extraction were carried out in the same manner as in Example 3.

The results showed that the mutant strain E.coli T10 containing FgGS could only synthesize the sesquiterpene compound (3) (Fig. 13c, Fig. 14), and confirmed by NMR data that it was synthesized with the EvVS synthesized monocyclic compound 2E-alpha. -cericerene structure is consistent.

Example 7 Synthesis of FgGS-derived Diterpenoids

To produce the diterpenoid compound, the two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transformed into E. coli BL21 (DE3) to obtain BL21(DE3)/pMH1/pFZ81, designated PS, and then pGB313 was transformed into the strain PS. In strain, strain T11 was obtained (Fig. 12). Subsequently, fermentation and product extraction were carried out in the same manner as in Example 3.

The results showed that the mutant strain E. coli T11 containing FgGS was able to synthesize 14 diterpenoids using GGPP as a substrate, and we purified and identified compound 5-10 (Fig. 13c, Fig. 14). The results show that the compound (5-10) is a novel class of skeleton compounds. Wherein the compound (5) is a quaternary diterpene compound of 5-5-5-5 ring; the compounds (6) and (8) are ternary diterpene compounds having 5-5-9 rings having different double arrow positions; Compound (7) is a quaternary diterpene compound of 5-5-7-4 ring; compounds (9) and (10) are 5-5-7-4 and 5-5-6-5 ring-free double bonds a quaternary sterol compound. It is worth noting that the first two 5-5 membered rings of these six compounds are their common structure. It shows that they have some common cyclization steps at the beginning of the synthesis, which is named GJ1012A–F.

Example 8 Synthesis of FgGS-derived monoterpenoids and sesquiterpene compounds

In order to produce monoterpene compounds and sesquiterpene compounds, GPP was synthesized based on FPPS with IPP and DMAPP as substrates, and then 1 molecule of IPP was used to generate FPP, and the two plasmids pMH1 and pFZ81 of the mevalonate pathway were simultaneously transferred. BL21(DE3)/pMH1/pFZ81 was obtained in E. coli BL21 (DE3) and designated as PS, and then pGB314 was transformed into strain PS to obtain strain T12 (Fig. 12). Subsequently, fermentation and product extraction were carried out in the same manner as in Example 3.

The results showed that the mutant strain E.coli T12 containing FgGS was able to synthesize four diterpenoids and three diterpenoids using FPP as a substrate (Fig. 13c, Fig. 14). The structure of two diterpenoids and the compound of FgMS product (11) is consistent with (23); the compounds (23), (43), and (44) are divalent quinone compounds of unknown structure. The three compounds synthesized are compounds (13) and (14) and linalool, respectively.

Example 9 Synthesis of AaTS-derived Diterpene Product Compound

AaTS is a newly discovered terpenoid synthase of the present invention derived from Alternaria alternata. AaTS was codon-optimized and ligated to vector pET28a by restriction enzyme site NdeI/EcoRI to obtain plasmid pGB136. The AaTS protein was subsequently purified as described in Example 1 and subjected to an in vitro reaction. The results of the in vitro reaction showed that AaTS was able to synthesize diterpene compounds using GGPP as a substrate (Fig. 15).

After demonstrating that it can synthesize diterpenoids, we amplified AaTS, GGPPS-Aa and pGB307 backbones with primers P48/49, P50/P51 and P52/P53, respectively, and assembled the above three fragments by Gibson method to obtain plasmids. pGB147 (Figure 11). Subsequently, we co-transformed pMH1, pFZ81 and pGB147 into E. coli BL21 (DE3) to obtain mutant strain T13 (Fig. 12), which was fermented and purified as described in Example 3.

By NMR analysis, we determined that the product synthesized by AaTS has the same structure as that of the product Traversiadiene isolated from Cercospora traversiana (Fig. 15), which is a precursor of Traversianal which has a good killing effect on molluscs. Stoessl A, Cole RJ, Abramowski Z, et al. Some Biological properties of traversianal, a strongly muluscicidal diterpenoid aldehyde from Cercospra traversiana [J]. Mycopathologia, 1989, 106 (1): 41-46.), the discovery of AaTS enables the production of Traversiadiene and its biosynthesis by combinatorial biosynthesis The mechanism is possible.

Example 10 Synthesis of FgAS-derived sesquiterpene product compound

Indole synthase 6 (FgAS, F. graminearum AJ1012 synthase) is an enzyme highly similar to indole synthase 1 (FgMS). It differs from FgMS in amino acid 65, the amino acid sequence of this site in FgMS is F, and the amino acid sequence of this site in FgAS is L. Using the same strategy, a second sesquiterpene novel skeleton compound (54) was detected in the diclofenium-producing strain containing FgAS by the method of Example 3.

Example 11 Compound Identification

Compound (1)

Figure 16 shows the spectrum of the compound (1), and the ¹ H NMR data indicates that the compound (1) has four single-peak methyl signals (Me-22, Me-23, Me-24, Me-25), 2 Bimodal methyl signals (Me-20 and Me-21), and 2 olefinic hydrogens (H-1 and H-18) (Table 2). ¹³ C NMR and heteronuclear single quantum correlation spectroscopy (HSQC) confirmed the presence of 25 carbon atoms, of which 3 sp ³ hybridized quaternary carbon atoms (C-6, C-11 and C-15), 2 sp ² hetero The quaternary carbon atoms (C-2 and C-19), 4 aliphatic methine groups, 2 enemethine groups, 8 methylene groups and 6 methyl groups. These data suggest that the compound (1) is a tetracyclic structure. The coupling relationship of ¹ H- ¹ H COSY is: H-20/H-3/H-4/H-5, H-7/H-21, H-10/H-14 and H-17/H- 18. The HMBC map shows that the related signals of methyl hydrogen are: Me-20 and C-2, C-3, C-4; Me-21 and C-3, C-6, C-7, C-8; Me -22 and C-1, C-11, C-12, C-14; Me-23 and C-13, C-14, C-15, C-16; Me-24 and C-18, C-19 , C-25; Me-25 and C-18, C-19, C-24. In addition, the HMBC map suggests a coupling relationship between H-5 and C-4, C-5, C-7; H-1 and C-3, C-6, C-11; H-18 and C-16. Therefore, the planar structure of the compound (1) is a tetracyclic sesquiterpene.

Compound (2)

Figure 17 shows the spectrum of the compound (2), and the ¹ H NMR data indicates that the compound (2) has 6 methyl signals (Me-20, Me-21, Me-22, Me-23, Me-24, Me- 25) and 4 olefinic hydrogens (H-2, H-6, H-9 and H-18) (Table 3). ¹³ C NMR and HSQC confirmed the presence of 25 carbon atoms, of which 2 sp ³ hybrids and 4 sp ² hybridized quaternary carbon atoms, 4 enemethine groups, 9 methylene groups and 6 methyl groups. These data suggest that the compound (2) is a bicyclic structure. The coupling relationship of ¹ H- ¹ H COSY is: H-1/H-2, H-5/H-6, H-8/H-9, H-16/H-17/H-18. The HMBC map shows that the related signals of methyl hydrogen are: Me-20 and C-2, C-3, C-4; Me-21 and C-6, C-7, C-8; Me-22 and C. -1, C-11, C-12; M-23 and C-13, C-14, C-15, C-16; Me-24 and C-18, C-19, C-25; Me-25 With C-18, C-19, C-24. In addition, the chemical shifts of C-20 and C-21 were 14.96 and 18, suggesting that the double bonds between C-2 and C-3, C-6 and C-7 are in the E configuration. Therefore, the planar structure of the compound (2) is a 11-6 membered bicyclic sesquiterpene.

Compound (3)

Figure 18 shows the spectrum of the compound (3), which is (2E)-α-cericerene. [α]

=+2.3(c 0.086,benzene). ¹ H NMR data indicated the presence of 6 methyl signals (Me-20, Me-21, Me-22, Me-23, Me-24, Me-25) and 5 olefins (Table 4). ¹³ C NMR and HSQC confirmed the presence of 25 carbon atoms, of which 5 sp ² hybridized quaternary carbon atoms (C-3, C-7, C-11, C-15, C-19), 1 aliphatic And 4 enemethyl groups, 8 methylene groups and 6 methyl groups. These data suggest that the compound (3) has a single ring structure. The coupling relationship of ¹ H- ¹ H COSY is: H-1/H-2, H-5/H-6, H-9/H-10, H-12/H-13/H-14, H- 16/H-17/H-18. The HMBC map shows that the related signals of methyl hydrogen are: Me-20 and C-2, C-3, C-4; Me-21 and C-6, C-7, C-8; Me-22 and C. -10, C-11, C-12; M-23 and C-14, C-15, C-16; Me-24 and C-18, C-19, C-25; Me-25 and C-18 , C-19, C-24. In addition, the HMBC map suggests that H-2 is coupled with C-14, C-4; H-6 and C-4, C-8; H-10 and C-8. Therefore, the compound (3) is a 14-membered monocyclic sesquiterpene.

Compound (4)

Figure 19 shows the spectrum of the compound (4), which is a colorless oily compound (R)-cembrene A. [α]

_{= -13.16 (c 0.053, CHCl 3} ). ¹ H NMR (400 MHz, deuterated chloroform) δ 5.19 (t, J = 7.4 Hz, 1H), 5.05 (t, J = 6.3 Hz, 1H), 4.98 (t, J = 6.5 Hz, 1H), 4.72 - 4.69 (m, 1H), 4.67–4.63 (m, 1H), 2.31–2.22 (m, 1H), 2.20–2.17 (m, 1H), 2.17–2.15 (m, 1H), 2.15–2.12 (m, 2H) ), 2.12–2.09 (m, 2H), 2.07–2.05 (m, 1H), 2.05–1.96 (m, 3H), 1.96–1.89 (m, 1H), 1.84–1.72 (m, 1H), 1.71–1.63 (m, 1H), 1.67 - 1.65 (m, 3H), 1.59 (s, 3H), 1.57 (s, 3H), 1.56 (s, 3H), 1.44 - 1.31 (m, 1H). ¹³ C NMR (101 MHz, cdCl ₃ ) δ 149.29, 134.79, 133.91, 133.43, 125.90, 124.05, 121.85, 110.11, 45.98, 39.41, 38.95, 33.98, 32.43, 28.21, 24.89, 23.76, 19.31, 18.00, 15.52, 15.30. HRMS (ESI) calculated for C ₂₀ H ₃₁ [MH] ⁺ : m/z 271.2420; m/z found: 271.2405. These data are consistent with previously reported (R)-cembrene A.

Compound (5)

Figure 20 shows the spectrum of the compound (5), and the ¹ H NMR data indicates that the compound (5) has four single-peak methyl signals (Me-17, Me-18, Me-19, Me-20), 1 Bimodal methyl signal (Me-16) and 1 olefinic hydrogen (H-6) (Table 5). ¹³ C NMR and HSQC confirmed the presence of 20 carbon atoms, of which 3 sp ³ hybridized quaternary carbon atoms (C-3, C-12, C-15), 1 sp ² hybridized quaternary carbon atom (C -7), 4 aliphatic methine groups, 1 enemethine group, 6 methylene groups and 5 methyl groups. These data suggest that the compound (5) is a tetracyclic structure. The coupling relationship of ¹ H- ¹ H COSY is: H-1/H-2/H-10/H-9/H-8, H-4/H-16. The HMBC map shows that the signals related to methyl hydrogen are Me-16 and C-3, C-4, C-5; Me-17 and C-3, C-6, C-7; Me-18 and C- 11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13, C-18; Me-20 and C-1, C-11, C-14, C-15. In addition, the HMBC map suggests that H-4 has a coupling relationship with C-2, C-6, and C-7. Therefore, the planar structure of the compound (5) is 5-5-5-5-membered tetracyclic diterpenes.

Compound (6)

Figure 21 shows the spectrum of the compound (6), and the ¹ H NMR data indicates that the compound (6) has five methyl signals (Me-16, Me-17, Me-18, Me-19 and Me-20), and 1 olefinic hydrogen (H-6) (Table 6). ¹³ C NMR and HSQC confirmed the presence of 20 carbon atoms, of which 2 sp ³ hybridized quaternary carbon atoms (C-12, C-15), 3 sp ² hybridized quaternary carbon atoms (C-2, C -3, C-7), 2 aliphatic methine groups, 1 enemethine group, 7 methylene groups and 5 methyl groups. These data suggest that compound (6) is a tricyclic structure. The coupling relationship of ¹ H- ¹ H COSY is: H-5/H-6, H-8/H-9. The HMBC map shows that the signals related to methyl hydrogen are Me-16 and C-2, C-3, and C-4; Me-17 and C-6, C-7, C-8; Me-18 and C. -11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13, C-18; Me-20 and C-1, C-11, C-14 , C-15. In addition, the HMBC map suggests that H-6 has a coupling relationship with C-5, H-9 and C-7, and H-10 with C-1, C-2, C-3, C-9 and C-11. Therefore, the planar structure of the compound (6) is 5-5-9-membered tricyclic dioxime.

Compound (7)

Figure 22 shows the spectrum of the compound (7), and the ¹ H NMR data indicates that the compound (7) has five methyl signals Me-16, Me-17, Me-18, Me-19, Me-20 (Table 7). . ¹³ C NMR and HSQC confirmed the presence of 20 carbon atoms, of which 3 sp ³ hybridized quaternary carbon atoms (C-3, C-12, C-15), 2 sp ² hybridized quaternary carbon atoms (C -6, C-7), 3 aliphatic methine groups, 7 methylene groups and 5 methyl groups. These data suggest that the compound (7) has a tetracyclic structure. The coupling relationship of ¹ H- ¹ H COSY is: H-1/H-2/H-10/H-11, H-4/H-5 and H-8/H-9. The HMBC map shows that the signals related to methyl hydrogen are Me-16 and C-2, C-3, C-4, C-6; Me-17 and C-6, C-7, C-8; Me- 18 with C-11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13, C-18; Me-20 and C-1, C-11, C-14, C-15. Therefore, the planar structure of the compound (7) is 5-5-7-4 membered tetracyclic diterpene.

Compound (8)

Figure 23 shows the spectrum of the compound (8). The ¹ H NMR results indicate that the compound (8) has four methyl groups (Me-17, Me-18, Me-19 and Me-20), and one terminal olefin. Hydrogen (H-16) (Table 8). ¹³ C NMR and HSQC confirm the presence of 20 carbon atoms, including two sp ³ hybridized quaternary carbon atom (C-12, C-15 ) and 2 an sp ² hybrid quaternary carbon atom (C-3, C -7), 1 methine group, 3 aliphatic methine groups, 1 olefinic group, 7 aliphatic methylene groups and 4 methyl groups. These data suggest that the compound (8) is a tricyclic structure. The coupling relationship of ¹ H- ¹ H COSY is: H-1/H-2, H-5/H-6, H-8/H-9/H-10. In addition, the HMBC map can be seen that the related signals of methyl hydrogen are Me-17 and C-6, C-7, C-8; Me-18 and C-11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13, C-18; Me-20 and C-1, C-11, C-14, C-15. In addition, the HMBC map suggests a coupling relationship between H-5 and C-4; H-10 and C-2 and C-11; H-16 and C-2, C-4. Therefore, the planar structure of the compound (8) is 5-5-9-membered tricyclic diterpenes.

Compound (9)

Figure 24 shows the spectrum of the compound (9). The ¹ H NMR results indicated that the compound (9) had five methyl signals Me-16, Me-17, Me-18, Me-19 and Me-20 (Table 9). . ¹³ C NMR and HSQC confirmed the presence of 20 carbon atoms, including 4 sp ³ hybrid quaternary carbon atoms (C-3, C-7, C-12 and C-15), 4 aliphatic methine groups, 7 methylene groups and 5 methyl groups. These data suggest that compound (9) is a tricyclic tertiary alcohol structure. The coupling relationship of ¹ H- ¹ H COSY is: H-1/H-2, H-4/H-5/H-6, H-8/H-9 and H-10/H-11. In addition, the HMBC map can see that the related signals of methyl hydrogen are Me-16 and C-2, C-3, C-4, C-6; Me-17 and C-6, C-7, C-8; Me-18 and C-11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13 and C-18; Me-20 and C-1, C- 11, C-14, C-15. In addition, the HMBC map suggests a coupling relationship between H-8 and C-2, C-8, C-9, C-10, and C-11. Therefore, the planar structure of the compound (9) is 5-5-7-4 membered tetracyclic diterpenes.

Compound (10)

Figure 25 shows the spectrum of the compound (10), and the ¹ H NMR results indicate that the compound (10) has five methyl groups (Me-16, Me-17, Me-18, Me-19 and Me-20), 1 A secondary alcohol H-6 (Table 10). ¹³ C NMR and HSQC confirmed the presence of 20 carbon atoms, including 4 sp ³ hybrid quaternary carbon atoms (C-3, C-7, C-12 and C-15), 4 aliphatic methine groups, 7 methylene groups and 5 methyl groups. These data suggest that compound (10) is a tetracyclic structure. The coupling relationship indicated by ¹ H- ¹ H COSY is: H-4/H-5/H-6, H-8/H-9/H-10, H-1/H-2. In addition, the HMBC map can be seen that the signals related to methyl hydrogen are Me-16 and C-2, C-3, C-4, C-7; Me-17 and C-3, C-6, C-7, C-8; Me-18 and C-11, C-12, C-13, C-19; M-19 and C-11, C-12, C-13, C-18; Me-20 and C- 1, C-11, C-14, C-15. In addition, the HMBC map suggests a coupling relationship between H-2 and C-10, H-10 and C-11. Therefore, the planar structure of the compound (10) is 5-5-6-5-membered tetracyclic diterpenes.

Compound (11)

Figure 26 shows the spectrum of the compound (11), which is a color oil trans-nerolidol. ¹ H NMR (400MHz, deuterochloroform) δ5.91 (dd, J = 17.3,10.8Hz , 1H), 5.21 (dd, J = 17.3,1.3Hz, 1H), 5.13 (t, J = 5.8Hz, 1H ), 5.10–5.05 (m, 1H), 5.06 (dd, J = 10.8, 1.3 Hz, 1H), 2.11–2.00 (m, 4H), 1.99–1.95 (m, 2H), 1.67 (s, 3H), 1.59 (s, 6H), 1.58 (m, 2H), 1.27 (s, 3H). ¹³ C NMR (101 MHz, cdCl 3 ) δ 145.02, 135.60, 131.46, 124.20, 124.17, 111.66, 73.52, 42.01, 39.69, 27.90, 26.62, 25.70, 22.71, 17.69, 16.01. HRMS (ESI) calculated for C ₁₅ H ₂₅ [M-OH] ⁺ : m/z 205.1951; m/z found: 205.1939.

Compound (12)

Figure 27 shows the spectrum of the compound (12) which is the

compound

2E, 6E-farnesol as a color oil. ¹ H NMR (400 MHz, deuterated chloroform) δ 5.42 (t, J = 7.0 Hz, 1H), 5.10 (q, J = 6.9 Hz, 2H), 4.15 (d, J = 6.9 Hz, 2H), 2.12 - 2.08 (m, 2H), 2.07 - 2.03 (m, 4H), 2.00 - 1.96 (m, 2H), 1.68 (s, 6H), 1.60 (s, 6H). ¹³ C NMR (101 MHz, cdcl3) δ 139.87, 135.35, 131.36, 124.26, 123.72, 123.24, 59.40, 39.67, 39.52, 26.68, 26.26, 25.70, 17.69, 16.28, 16.00. HRMS (ESI) calculated for C ₁₅ H ₂₅ [M-OH] ⁺ : m/z 205.1951; m/z found: 205.1940.

Compound (53)

Figure 28 shows the spectrum of Compound (53), ¹ H NMR data suggest that compound (53) there are three singlet methyl signals (Me-17, Me-18 , Me-20), 1 methyl doublets Base signal (Me-19), and 2 olefinic hydrogens (H-9 and H-16) (Table 11). ¹³ C NMR and heteronuclear single quantum correlation spectroscopy (HSQC) confirmed the presence of 20 carbon atoms with one sp ³ hybrid quaternary carbon (C-11) and two sp ² hybrid quaternary carbon atoms ( C-7 and C-14), 5 aliphatic methine groups, 1 enemethine group, 6 aliphatic methylene groups, 1 exo-methylene group and 4 methyl groups. These data suggest that compound (53) is a tetracyclic structure. The coupling relationship of ¹ H- ¹ H COSY is: H-1/H-2, H-3/H-19 and H-8/H-9. The HMBC map shows that the related signals of methyl hydrogen are: Me-17 and C-14, C-15 and C-16; Me-18 and C-6, C-7 and C-8; M-19 and C -2, C-3 and C-4; Me-20 and C-1, C-10, C-11 and C-12. In addition, the HMBC map suggests H-1 and C-2, C-6, C-10; H-3 and C-5, C-6; H-5 and C-6, C-7; H-8 and C- 10; H-10 and C-15; there is a coupling relationship between H-13 and C-15. Therefore, the planar structure of the compound (53) is a tetracyclic sesquiterpene.

Compound (54)

Figure 29 shows the spectrum of the compound (54), and the ¹ H NMR data indicates that the compound (54) has 6 methyl signals (Me-20, Me-21, Me-22, Me-23, Me-24, Me- 25). ¹³ C NMR, heteronuclear single quantum correlation spectroscopy (HSQC) and DEPT 135° confirmed the presence of 25 carbon atoms with 3 sp ³ hybrid quaternary carbon atoms (C-11, C-14, C-17), 3 sp ² hybrid quaternary carbon atoms (C-2, C-3, C-7), 3 aliphatic methine groups, 1 ene methine group, 9 aliphatic methylene groups, and 6 Methyl. These data suggest that compound (54) is a tetracyclic structure. The coupling relationship of ¹ H- ¹ H COSY is: H-4/H-5/H-6, H-8/H-9 and H-15/H-16. The HMBC map shows that the related signals of methyl hydrogen are: Me-20 and C-2, C-3 and C-4; Me-21 and C-6, C-7 and C-8; M-22 and C -1, C-10,

C-11 and C-12; Me-23 and C-13, C-14, C-15 and C-19; Me-24 and C-16, C-17, C-18 and C-25; Me- 25 with C-16, C-17, C-18 and C-24. In addition, the HMBC map suggests a coupling relationship between H-1 and C-2, C-3; H-9 and C-11; H-18 and C-19. Therefore, the planar structure of the compound (54) is a 5-8-6-6 membered tetracyclic sesquiterpene.

Table 2 Nuclear magnetic data of compound (1)

Table 3 Nuclear magnetic data of compound (2)

Table 4 Nuclear magnetic data of compound (3)

Table 5 Nuclear magnetic data of compound (5)

Table 6 Nuclear magnetic data of compound (6)

Table 7 Nuclear magnetic data of compound (7)

Table 8 Nuclear magnetic data of compound (8)

Table 9 Nuclear magnetic data of compound (9)

Table 10 Nuclear magnetic data of compound (10)

Table 11 Nuclear magnetic data of compound (53)

Table 12 Nuclear magnetic data of compound (54)

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms is not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, various embodiments or examples described in the specification, as well as features of various embodiments or examples, may be combined and combined.

Although the embodiments of the present invention have been shown and described, it is understood that the above-described embodiments are illustrative and are not to be construed as limiting the scope of the invention. The embodiments are subject to variations, modifications, substitutions and variations.

Claims

An indole synthase, characterized in that the catalytic substrate of the indole synthase is a compound having 10 to 25 carbon atoms.
The indole synthase according to claim 1, wherein the catalytic substrate is selected from one of the following:

Geranyl pyrophosphate

Farnesyl pyrophosphate

Geranylgeranyl pyrophosphate;

Geranyl-based farnesyl pyrophosphate.
The indole synthase according to claim 1, wherein the indole synthase has the amino acid sequence of any one of SEQ ID NOS: 1 to 6.
A nucleic acid molecule encoding the indole synthase of claim 1

Optionally, the nucleic acid molecule has the nucleotide sequence set forth in any one of SEQ ID NOs: 7-12.
A construct comprising the nucleic acid molecule of claim 4.
A recombinant cell characterized by comprising:

a first nucleic acid molecule encoding a purine synthase,

Optionally, the steroid synthase has the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 6,

Optionally, the first nucleic acid molecule has the nucleotide sequence set forth in any one of SEQ ID NOs: 7-12.

Optionally, the recombinant cell further comprises:

a second nucleic acid molecule, the second nucleic acid molecule being selected from at least one of the following:

AntoB gene or idi gene derived from E. coli XL1-blue;

The erg13 gene, tHMG1 gene, erg12 gene, erg8 gene or mvd1 gene derived from Saccharomyces cerevisiae INVSC1.
Use of the steroid synthase according to any one of claims 1 to 3 or the nucleic acid molecule of claim 4 or the construct of claim 5 or the recombinant cell of claim 6 for synthesizing terpenoids.
The use according to claim 7, wherein the synthesis is carried out in a host cell, and the catalytic substrate of the steroid synthase is by overexpressing at least one of the following genes in a host cell. acquired:

AntoB gene or idi gene derived from E. coli XL1-blue;

The erg13 gene, tHMG1 gene, erg12 gene, erg8 gene or mvd1 gene derived from Saccharomyces cerevisiae INVSC1.
The use according to claim 7, wherein the quinone compound has a structure of one of the following:
A method of synthesizing a terpenoid according to claim 9, comprising:

The recombinant cell of claim 6 is cultured under conditions suitable for expression of the terpenoid to obtain a culture product;

The quinone compound is isolated from the culture product.